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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q2531/00—Reactions of nucleic acids characterised by
  • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
  • C12Q2531/113—PCR
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to a method for evaluating and categorizing the potential for ocular irritation of chemicals.
• the Draize Test is an invasive toxicological test based on an animal testing protocol developed in 1944 by John H. Draize and Jacob M. Spines, toxicologists of the Food and Drug Administration (FDA).
• the test consists in testing a product in the eye of an animal in order to evaluate its ocular safety.
• the chemicals are classified according to a globalized system and put in place by the United Nations known as the United Nations Globally Harmonized Sytem and concerning the properties of eye irritation, the products are classified into several categories.
• Category 1 is for damage causing products that are not reversible after 21 days
• category 2A for irreversible eye irritants before 21 days
• category 2B for medium irritating products for the eyes. reversible before 21 days.
• Non-irritating products are not classified.
• European legislation combines categories 2A and 2B into a single category 2 described as reversibly irritating to the eyes.
• This method comprises the in vitro cultivation of an epidermal model, the deposition of the product to be studied on cultured epidermis model, and the quantification of the number of surviving living cells in order to evaluate the ocular discomfort or the irritating potential of it.
• Such a process, developed in replacement of animal experimentation has the disadvantage of being long and expensive and not very discriminating.
• this method is not an integral replacement method, since it does not reliably detect the entire range of intermediate irritations.
• the inventors have demonstrated that the response following the action of an irritating substance is directly on a corneal epithelium reconstructed in vitro, and that the degree of irritation, and the qualification of this irritation of a molecule, can be determined through the use of biomarkers specific to eye irritation. It is notable to note, and it is a surprising feature of the present method, that the molecular markers employed have never been described or disclosed in connection with eye irritation.
• the inventors have demonstrated that an in vitro reconstructed corneal epithelium is a sufficient model to reveal specific biomarkers of ocular irritation in humans, and that these biomarkers also make it possible to predict the degree of irritation and therefore to classify in a predictive manner the products tested according to the 3 categories as indicated above.
• the present invention relates to a method for evaluating the ocular irritation potential of a test compound, comprising the steps of: a) contacting a test compound with a reconstructed corneal sample in vitro; b) measuring the expression of at least one gene selected from the group consisting of: HSP90AA1, COL7A1, NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4, DDIT3, MMP3, HAS1, IRF1, CYR61.
• the method according to the present invention may further comprise a step c) of determining an eye irritation index of a test compound.
• the method also comprises a step d) of categorizing said compound as having an ocular irritation potential according to the value of the ocular irritation index obtained.
• the method according to the present invention is an in vitro method.
• the term "in vitro reconstructed corneal sample” refers to a sample of corneal epithelial cells cultured in a defined culture medium or any model using human squamous epithelial cells and having a morphology similar to the human cornea, such as in vitro corneal models of the type marketed under the trademark EpiOcular®.
• this "corneal sample reconstructed in vitro" is a sample comprising, or constituted by immortalized corneal epithelial cells, cultured in a defined culture medium and placed in a thin layer on a synthetic membrane at the water-air interface.
• said immortalized corneal epithelial cells are human cells.
• this "corneal sample reconstructed in vitro" is a HCE SkinEthic® sample marketed by the company Episkin (Lyon, France) which is a reconstructed cornea epithelium consisting of human corneal keratinocytes, particularly cells transformed for to be made immortal.
• the contacting of the test compound in step a) is carried out with the test compound in the solid form, for example in powder form.
• the contacting of the test compound in step a) is carried out with the test compound in the liquid form.
• the measurement of the expression of said at least one gene of step b) makes it possible to determine the level of expression of said gene.
• the test compound may be a compound of varied nature, structure and origin, including a biological compound, a chemical compound, synthetic compound, etc.
• the test compound can be any product that is in an isolated form or in admixture with other products.
• the test compound can be defined in terms of structure and / or composition or be defined functionally.
• the test compound may, for example, be an isolated and structurally defined product, an isolated product of indefinite structure, a mixture of known and characterized products or a composition comprising one or more products. One or more compounds can thus be tested, in admixture or separately.
• the present invention is particularly suitable for the identification of a large number of compounds. This simple and efficient screening can be accomplished in a very short time. In particular, the described methods can be partially automated, thus enabling efficient and simultaneous screening of various and diverse compounds, either as a mixture or in separate form.
• the level of expression of said gene is evaluated by measuring the level of expression of the polypeptide encoded by said gene or a fragment thereof, or by measuring the level of expression of the gene. mRNA of said gene or fragment thereof.
• the expression of said at least one gene is performed by analyzing the expression of mRNA transcripts or mRNA precursors, such as RNA. native, said gene.
• Said assay can be performed by preparing the mRNA / cDNA of cells from a biological sample of a patient, and hybridizing the mRNA / cDNA with a reference polynucleotide.
• the mRNA / cDNA prepared can be used in a hybridization or amplification assay which includes, but is not limited to, Southern and Northen analyzes, polymerase chain reaction (PCR) assays, such as quantitative PCR (Taqman ) and the use of "probes arrays" such as GeneChip® DNA templates (AFFYMETRIX).
• PCR polymerase chain reaction
• the analysis of the expression of the level of transcribed mRNA of the said at least one gene involves a nucleic acid amplification process, such as, for example, RT-PCR (experimental embodiment described in US Pat. No. 4,683,202), the ligase chain reaction (BARANY, Proc Natl Acad Sci USA, vol.88, p: 189-193, 1991), self sustained sequence replication (GUATELLI) et al., Proc Natl Acad Sci USA, vol.87, p: 1874-1878, 1990), the transcriptional amplification system.
• RT-PCR experimental embodiment described in US Pat. No. 4,683,202
• the ligase chain reaction BARANY, Proc Natl Acad Sci USA, vol.88, p: 189-193, 1991
• GUILLI self sustained sequence replication
• the method according to the present invention comprises a further step of amplification of the mRNA or cDNA of said gene, the complementary sequence thereof or a fragment thereof.
• the amplification primers are defined as a pair of nucleic acid molecules that can match the 3 'and 5' regions of a gene, specifically (positive and negative strands, or conversely) and frame a short region of said gene.
• the amplification primers have a length of 10 to 30 nucleotides and allow the amplification of a region of a length of between 50 and 200 nucleotides.
• the measurement of the expression of said at least one gene is performed by measuring the level of expression of the polypeptide encoded by said gene.
• Said assay can be performed using an antibody (eg, a radiolabeled antibody, labeled with a chromophore, a fluorophore, or an enzyme), an antibody derivative (eg an antibody conjugated to a substrate or a protein or a ligand of a protein of a ligand / protein pair (e.g., biotin-streptavidin)) or an antibody fragment (e.g., a single chain antibody, a hypervariable domain of an isolated antibody, etc.) that specifically binds to the polypeptide encoded by said gene.
• an antibody eg, a radiolabeled antibody, labeled with a chromophore, a fluorophore, or an enzyme
• an antibody derivative eg an antibody conjugated to a substrate or a protein or a ligand of a protein of a
• Said assays can be performed by many techniques within the abilities of those skilled in the art including, but not limited to, immunoassays based on the use of enzyme activity ("enzyme immunoassay” EIA), immunoassay based on the use of radioactive isotopes (RIA), the analysis by Western blot and ELISA (enzyme linked immunoabsorbent assay) assays.
• enzyme immunoassay EIA
• RIA radioactive isotopes
• Western blot and ELISA enzyme linked immunoabsorbent assay
• polypeptide means a sequence comprising at least two amino acids, and the terms “polypeptide”, “peptide” and “protein” may be used interchangeably.
• mRNA or cDNA fragment is meant a sequence of at least 50 nucleic acids, for example at least 100 or 150 nucleic acids, preferably at least 200 nucleic acids. as an example of at least 250 or 350 nucleic acids, and particularly preferably a polypeptide of at least 400 nucleic acids.
• fragment of the polypeptide is meant a sequence of at least 50 amino acids, for example at least 100 or 150 amino acids, preferably at least 200 amino acids, for example at least 250 or 350 amino acids, and particularly preferably a polypeptide of at least 400 amino acids.
• step b) comprises measuring the expression of at least two genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4, DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least two genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in which step b) comprises measuring the expression of at least three genes chosen from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4 , DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least three genes chosen from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R
• step b) comprises measuring the expression of at least four genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4 , DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least five genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4 , DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least six genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4, DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least six genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A
• step b) comprises measuring the expression of at least seven genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4 , DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least eight genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4 , DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least nine genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4 , DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least ten genes selected from the group consisting of: HSP90AA1, COL7A1, NOS3, MMP8, CASPI, ELN, IL-23R, DLK1,
• step b) comprises measuring the expression of at least eleven genes chosen from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4 , DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least twelve genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4 , DDIT3, MMP3, HAS1, IRF1, CYR61.
• step b) comprises measuring the expression of at least thirteen genes selected from the group consisting of: HSP90AA1, COL7A1 , NOS3, MMP8, CASP1, ELN, IL-23R, DLK1, CLEC4D, IL-24, CCL22, SLIT2, ICAM2, MUC13, HSPA1A, FSHR, IL-1R2, ALB, CCND1, CXCL1, CXCR1, KL, COL6A2, MUC4 , DDIT3, MMP3, HAS1, IRF1, CYR61.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) comprises measuring the expression of at least one gene selected from the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL-24, SLIT2, HSPA1A, FSHR, IL-1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) comprises measuring the expression of at least two genes selected from the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL-24, SLIT2, HSPA1A, FSHR, IL-1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) comprises measuring the expression of at least three genes selected from the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL-24, SLIT2, HSPA1A, FSHR, IL-1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) comprises measuring the expression of at least four genes selected from the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL-24, SLIT2, HSPA1A, FSHR, IL-1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) comprises measuring the expression of at least five genes selected from the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL24, SLIT2, HSPA1A, FSHR, IL1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) comprises measuring the expression of at least six genes selected from the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL24, SLIT2, HSPA1A, FSHR, IL1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) comprises measuring the expression of at least seven genes selected from the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL-24, SLIT2, HSPA1A, FSHR, IL1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) includes the measurement of the expression of at least eight genes selected from the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL24, SLIT2, HSPA1A, FSHR, IL1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) comprises measuring the expression of at least nine genes selected from the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL24, SLIT2, HSPA1A, FSHR, IL1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in liquid form, in which the step b) comprises measuring the expression of the genes of the group consisting of: HSP90AA1, CASP1, DLK1, CLEC4D, IL24, SLIT2, HSPA1A, FSHR, IL1R2 and CCND1.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of at least one gene selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in the form of solid, in which step b) comprises measuring the expression of at least two genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of at least three genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of at least four genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of at least five genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound with step a) is carried out with the test compound in solid form, wherein step b) comprises measuring the expression of at least six genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of at least seven genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of at least eight genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of at least nine genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, particularly when the contacting of the test compound in step a) is carried out with the test compound in solid form, wherein step b) comprises measuring the expression of at least ten genes selected from the group consisting of by: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of at least eleven genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of at least twelve genes selected from the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• said method makes it possible to evaluate the ocular irritation potential of a test compound, in particular when the contacting of the test compound in step a) is carried out with the test compound in solid form, in which the step b) comprises measuring the expression of the genes of the group consisting of: IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• the test compound contacted with the reconstructed corneal sample in vitro is in liquid or solid form. When the test compound is liquid, it can be used pure or diluted.
• the compound may be diluted in a physiologically acceptable solvent, such as a PBS (Phosphate Buffer Saline) buffer for example, and is therefore present in a mass concentration of between 0.1% and 100%.
• a physiologically acceptable solvent such as a PBS (Phosphate Buffer Saline) buffer for example.
• PBS Phosphate Buffer Saline
• Step c) of the method comprises determining an eye irritation index of the compound.
• the determination of the ocular irritation index of the compound comprises assigning an overexpression threshold value to each gene whose expression is measured.
• the overexpression threshold value corresponds to a factor for increasing the expression of the gene during contacting with the test compound with respect to the expression of said gene during contacting with a control.
• the method according to the present invention further comprises a step of comparing the level of expression of said gene with a reference value.
• This reference value can serve as a positive and / or negative control.
• a positive control can for example be performed by comparing the level of expression of said at least one gene in the presence of the test compound with the level of expression of said at least one gene in the presence of a compound known as ocular irritant.
• the level of expression of said at least one gene in the presence of the test compound is greater than or equal to the level of expression of said at least one gene in the presence of a compound whose irritant potential is known, it can be concluded the irritating potential of said compound.
• a negative control can be carried out in the absence of the test compound or in the presence of a compound known to be non-irritating, such as, for example, olive oil, 1, 9-decadiene (CAS # 1647-16-1), triclobarban (case # 101-20-2) or the buffer in which the test product is dissolved or diluted when used in the method.
• a compound known to be non-irritating such as, for example, olive oil, 1, 9-decadiene (CAS # 1647-16-1), triclobarban (case # 101-20-2) or the buffer in which the test product is dissolved or diluted when used in the method.
• a test compound has an ocular irritant potential if overexpression of said gene is observed with respect to its level of expression in the absence of said test compound.
• overexpression is meant a significantly higher level of expression of said gene relative to its normal expression level.
• overexpression is understood to mean a level of expression in a biological sample which is greater than at least 20% of the normal level of expression of said gene (i.e., 1.2 times more), preferably greater than at least 50% of the normal level of expression of said gene (that is to say 1.5 times more), and particularly preferably at least 90% higher than the normal level of expression of said gene (c ie 1.9 times more).
• the "level of expression in the absence of said test compound” or "normal level” is the level of expression of said gene in a control sample potentially corresponding to the biological sample of a tissue not exhibiting a reaction. irritation or, preferably, the average level of expression of said gene in different control samples not exposed to the test compound.
• step b) is carried out between 2 and 24 hours after step a), more preferably between 4 and 18 hours after step a), particularly preferably between 5 and 7 hours after step a) and even more preferably 6 hours after step a).
• the control having a normal level of expression consists in bringing the reconstructed corneal sample in vitro into contact with a non-irritating physiologically acceptable liquid, such as for example the liquid in which is dissolve or dilute the test compound, for example a buffer, more particularly for example a PBS buffer.
• a non-irritating physiologically acceptable liquid such as for example the liquid in which is dissolve or dilute the test compound, for example a buffer, more particularly for example a PBS buffer.
• the threshold value indicating a significant overexpression of the gene whose expression is measured may be between 1.1 and 10, more particularly between 1.1 and 7, more particularly between 1.4 and 6, more particularly between 2 and 5. more particularly between 2 and 4.
• the preferred genes are selected from the group consisting of at least, or consisting of, HSP90AA1, CASP1, DLK1, CLEC4D, IL-24, SLIT2, HSPA1A, FSHR, IL-1R2 and CCND1.
• HSP90AA1 the overexpression threshold value is between 1.1 and 2, more particularly 1.4.
• the overexpression threshold value is between 1.1 and 2, more particularly about 1.5.
• the overexpression threshold value is between 3 and 8, more particularly about 5, more particularly 5.25.
• the overexpression threshold value is between 1.1 and 4, more particularly about 3.6.
• the overexpression threshold value is between 3 and 8, more particularly about 6.2.
• the overexpression threshold value is between 1.1 and 2, more particularly about 1.3.
• the overexpression threshold value is between 1.1 and 5, more particularly about 3.
• the overexpression threshold value is between 1.1 and 4, more particularly about 2.6.
• the overexpression threshold value is between 1.1 and 2, more particularly about 1.3.
• the overexpression threshold value is between 1.1 and 2, more particularly about 1.5.
• the preferred genes are selected from the group consisting of at least, or consisting of, IL- 24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13 and MUC4.
• the overexpression threshold value is between 1.5 and 15, more particularly between 2 and 10, more particularly 2.3.
• the overexpression threshold value can also be between 5 and 15, more particularly 10.
• the overexpression threshold value is between 1.5 and 3, more particularly about 1.9.
• the overexpression threshold value is between 1.5 and 3, more particularly about 5, more particularly 2.
• the overexpression threshold value is between 1.1 and 2.5, more particularly about 1.4.
• the overexpression threshold value is between 2 and 8, more particularly about 4.
• the overexpression threshold value is between 1.5 and 5, more particularly about 3.
• the overexpression threshold value is between 1.1 and 2, more particularly about 1.3.
• the overexpression threshold value is between 1.4 and 3, more particularly about 2.
• the overexpression threshold value is between 1.5 and 3.5, more particularly about 2.8.
• the overexpression threshold value is between 3 and 5, more particularly about 4.
• the overexpression threshold value is between 1.1 and 2, more particularly about 1.4.
• the overexpression threshold value is between 1.4 and 3, more particularly about 1.8.
• the overexpression threshold value is between 2 and 4, more particularly about 2.5.
• the significant overexpression threshold value can, for each gene, be evaluated and determined easily by a person skilled in the art.
• the determination of the eye irritation index includes the assignment of a value of weight to each gene if the overexpression threshold value of said gene is reached following the measurement of the expression.
• this gene is assigned a weight value that may be different or identical depending on the gene.
• This weight value can take discrete or continuous values, preferably discrete values ranging between 1 and 10, namely chosen from the group consisting of the values 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. More particularly, the discrete values may be selected from the group consisting of values 1, 2, 3, 4, 5. More particularly, said discrete values may be 1 or 2.
• the weight value may be limited to two values, 1 or 2, which value will be attributed to the gene when its significant overexpression threshold is reached or exceeded.
• the choice to assign a higher or lower weight value depends on the nature of the gene considered and its involvement in the cellular response to stress, which in the metabolic pathway of interleukin, which in the inflammatory process, in which the regulation of cell growth, which in healing, which in cell remodeling, for example but also the overexpression threshold value of the gene.
• the weight value can take a value chosen between 1 and 2.
• HSP90AA1 the weight value can be 2.
• CASP1 the weight value can be 2.
• DLK1 the weight value can be 2.
• CLEC4D the weight value can be 2.
• IL24 the weight value can be 1.
• SLIT2 the weight value can be 1.
• HSPA1A the weight value can be 2.
• FSHR the weight value can be 2.
• IL-1R2 the weight value can be 1.
• CCND1 the weight value can be 2.
• the weight value can take a value chosen between 1 and 4.
• the weight value can be 1.
• the weight value can be 4.
• the weight value can be 4.
• the weight value can be 2.
• the weight value can be 1.
• the weight value can be 2.
• HAS1 the weight value can be 2.
• CYR61 the weight value can be 1.
• IL-1R2 the weight value can be 4.
• CLEC4D the weight value can be 2.
• ICAM2 the weight value may be 4.
• CASP1 the weight value may be 2.
• MUC13 the weight value may be 3.
• MUC4 the weight value may be 4.
• the eye irritation index is determined by the addition of the weight values of the genes whose expression exceeds the overexpression threshold value.
• the method according to the invention may also comprise a step of categorizing the test compound comprising assigning an irritation category to the test compound as a function of the value of the ocular irritation index obtained.
• the method according to the invention thus provides the skilled person with a predictive method for classifying a compound according to its ocular irritation potential according to the European classification, namely non-irritating, reversible irritant or non-reversible irritant.
• the results set forth in the experimental part of the present application demonstrate this advantage of the invention and the superiority of this method over existing ones is evident from these results.
• the reconstructed human corneal epithelium model (SkinEthic TM HCE) was purchased from Episkin in Lyon.
• the model consists of immortalized human corneal epithelial cells cultured in a defined medium at the air-liquid junction.
• the tissue structure obtained is a multi-layer epithelium similar to the natural tissue structure of the human cornea comprising 5 -7 layers for a surface of 0.5 cm 2 . Polyhedral cells and winged cells are also present.
• the tissue also includes specific ultra-structures such as intermediate filaments, mature hemimmunosomes and desmosomes. Cytokeratin 65 kD (K3) was also detected (Nguyen et al., 2003).
• the tissues are shipped on a semi-solid layer of agarose culture medium. On reception, the tissues are transferred to maintenance medium (1 ml / well) in 6-well plates and incubated in a humidifier at 37 ° C., 5% CO 2 . The fabrics are used 24h later. Chemical products
• Liquid products are tested for solubility in PBS (phosphate buffered saline) buffer or in olive oil.
• PBS phosphate buffered saline
• 100 ⁇ l of the product to be tested is mixed with 200 ⁇ l of PBS or olive oil.
• the sample is mixed on a vortex. Turbidity and eventual phase separation are evaluated by the eye.
• the products are tested pure or diluted to 30%. Products that are not soluble in PBS or olive oil can not be tested at 30%.
• the procedure for applying liquid products to tissues has been optimized as follows: the chemicals are tested at two concentrations 100% and 30%.
• the tissue surface is moistened by the addition of 20 ⁇ PBS at 37 ° C and incubation for 10 min at 37 ° C / 5% CO 2 ⁇
• the corneal epithelia are then treated topically with 50 ⁇ 2 ⁇ l of the tested product (which corresponds to 100 ⁇ l / cm 2) and then incubated for 10 min at room temperature.
• the tissues are then washed with sterile PBS (2 X 25 ml) at 37 ° C. PBS is deposited on the edge of the insert (not directly on the fabric) to create a soft vortex that removes the chemical.
• the tissues on their inserts are then "drowned" in 5 ml of maintenance medium at room temperature for 30 minutes in order to remove the maximum remaining product on the surface of the fabric.
• the medium is then removed by gently tapping the insert on absorbent paper and 50 ⁇ of maintenance medium is added at 37 ° C.
• the inserts are then incubated for 6 hr at 37 ° C / 5% CO 2 .
• the procedure for applying solid (powdered) products to fabrics has been optimized as follows. At first the products are reduced in the finest powder possible by means of a mortar. Chemicals are tested at a single dose. In summary, the tissue surface is wetted by the addition of 20 ⁇ PBS at 37 ° C and incubation for 10 min at 37 ° C / 5% CO 2 . The corneal epithelia are then treated topically with 30 ⁇ 2 mg (representing 60 mg / cm 2 ) and then incubated for 30 minutes at room temperature. The tissues are then washed with sterile PBS (2 X 25 ml) at 37 ° C. PBS is deposited on the edge of the insert (not directly on the fabric) to create a soft vortex that removes the chemical.
• the tissues on their inserts are then "drowned” in 5 ml of maintenance medium at room temperature during 30 minutes to remove the maximum remaining product on the surface of the fabric.
• the medium is then removed by gently tapping the insert on absorbent paper and 50 ⁇ of maintenance medium is added at 37 ° C.
• the inserts are then incubated for 6 hr at 37 ° C / 5% CO 2 .
• RNAs The method for purifying total RNAs has been described by Cottrez et al., 2015.
• corneal tissues are retrieved with forceps and placed in tubes for rapid freezing in liquid nitrogen.
• the RNAs are then extracted by the technique of Qiazol (Qiagen, Courtaboeuf, France) with a kit "RNeasy Mini Kit” according to the manufacturer's instructions.
• the tissues are placed in 1 ml of Qiazol and homogenized using TissueLyser II (Qiagen, Courtaboeuf, France) with 2 steel balls. After centrifugation the supernatant is collected and 0.2 ml of bromochloro propane (Sigma, France) are added, then the whole is mixed vigorously.
• the homogenate is centrifuged at 12,000 g for 15 minutes at 4 ° C.
• the upper phase (aqueous phase) is added to 600 ⁇ l of 70% ethanol and immediately mixed by pipetting.
• the mixture is transferred to an RNeasy spin column placed on a 2 ml collection tube and RNA is collected according to the manufacturer's instructions (Qiagen, Courtaboeuf, France). Quantitative RT-PCR analysis
• the quantitative RT-PCR procedure has been described by Cottrez et al., 2015.
• the transcription of the total RNA is carried out with 1 ⁇ g of total RNA in final volume of 20 ⁇ l using Random Primers. (Invitrogen, France) and the "SuperScript III Reverse Transcriptase” (Invitrogen, France) according to the manufacturer's instructions.
• the quantitative RT-PCR uses a reactive PCR mixture: the SYBR Green real time Master PCR Mix (ROCHE, France) with 0.4 ⁇ l of each nucleotide primer in a final volume of 25 ⁇ l.
• the reaction is carried out in an LC480 System (ROCHE, France).
• the amplification program comprises a cycle at 95 ° C for 1 min, followed by 40 cycles with denaturation at 95 ° C for 15s, a hybridization and amplification phase at 60 ° C for 15s, followed by phase x final elongation at 72 ° C for 40s.
• the relative amount of each transcript is normalized to the average amount of expression of "housekeeping" genes (Glucuronidase ⁇ -GUSB, vacuolar ATPase-ATP6V0E1, H2A Histone Family, Member Y-H2AFY, Glucose-6-Phosphate Dehydrogenase- G6PD and "non-POU domain containing, octamer-binding" -NONO).
• housekeeping genes Glucuronidase ⁇ -GUSB, vacuolar ATPase-ATP6V0E1, H2A Histone Family, Member Y-H2AFY, Glucose-6-Phosphate Dehydrogenase- G6PD and "non-POU domain containing, octamer-binding" -NONO).
• the gene expression rate is measured by an absolute quantization analysis method using the method based on the maximum of the second derivative developed by Roche.
• the relative increase rate fold increase is then calculated relative to the treated tissues with PBS alone.
• LU Liquid Irritation Index
• Each overexpressed gene (or each threshold exceeded for IL-24) then receives a fixed value of respectively: 1; 4; 4; 2; 1; 2; 2; 1; 4; 2; 4; 2; 3 and 4.
• An eye irritation index for solid substances (SU: Solid Irritation Index) is then calculated by adding the values assigned to each overexpressed gene to a maximum value of 36.
• SU Solid Irritation Index
• the prediction and classification model works as follows. Each product is tested at 60 mg / cm 2 (see above). If SU ⁇ 20 the tested product is classified as "Cat I”. If ⁇ SU ⁇ 20 the product tested is classified as "Cat 2". If SU ⁇ 10 the tested product is classified as "No-Cat".
• a non-irritant 1, 9-decadiene (CAS # 1647-16-1) 100% tested, and a Category 2 product: 2-methyl-1-pentanol (CAS # 105-30-6) tested at 100% and 30%.
• the test is considered valid if the LU values obtained for the 1, 9-decadiene tested at 100% and the 2-methyl-1-pentanol tested at 30% are less than 10 and the LU for the 2-methyl- l-pentanol 100% tested is> 10.
• a non-irritant triclobarban (case # 101-20-2) and a Category 2 product: naphthalene dione (case # 83-56-7).
• the test is considered valid if the value of SU obtained for the triclobarban is ⁇ 10 and if the value obtained for naphthalene dione is between 10 and 20.
• the list of selected genes for solids includes: (IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2, CLEC4D, ICAM2, CASP1, MUC13, MUC4, Figure 2).
• the SU has been developed in two stages. We first selected a threshold value for each gene to show significant overexpression.
• Figure 1 A, B, C, D Gene expression analysis in 3D reconstructed human corneal tissue treated with various irritant and irritant non-irritant liquid chemicals.
• the value of the fold increase rate for the 29 genes indicated was represented for 4 category 1 chemicals (black bar, in order from left to right lactic acid, methyl thioglycolate, sodium lauryl sulfate (15%) , Benzalkonium chorid (10%)), 4 category 2 chemicals (gray bars, in the order from left to right alpha hexyl cinnamaldehyde, acetone, methyl ethyl ketone, 3-chloro propane nitrile) and 4 unclassified products (No. category, white bars in order from left to right: 1,9 decadiene, Glycerol, Tween 20, 2,4 pentanediol).
• genes used for the example are HSP90AA1, CASP1, DLK1, CLEC4D, IL-24, SLIT2, HSPA1A, FSHR, IL-1R2 and CCND1.
• Figure 2 A and B Gene expression analysis in 3D reconstructed human corneal tissue treated with various irritant and irritant non-irritant solid chemicals.
• the value of the fold increase rate for the 29 genes indicated was represented for 5 category 1 chemicals (black bar, in order from left to right, 2 hydroxyisobutyric acid, Promethazine Hydrochloride, Sodium oxalate, 2 , 5-dimethyl heanediol, 1-naphthalene acetic acid), 2 chemicals of category 2 (gray bars in order from left to right, Naphthalene diol, Camphene) and 8 unclassified products (No category, white bars in order from left to right Triclocarban, Methylenebis benzotriazol tetramethylbutyl phenol, Pyrimetranyl, Myristil myristate, 4,4'-methylene bis- (2,6-di-tert-butylphenol), 4-bromophenetol, potassium tetrafluoroborate).
• the genes selected for the example are IL-24, IL-23R, DDIT3, MMP8, DLK1, HAS1, CYR61, IL-1R2 CLEC4D, ICAM2, CASP1, MUC13, MUC4.
• Table 2 List of genes analyzed by RT-PCR
• Table 3 Results of the analysis of the 39 liquid products tested by the eye irritation test (IOT) and comparison with the results obtained by the Draize test
• Table 5 Confusion matrix analysis of the prediction ability of the ocular irritation test to be separated into 3 classes of irritation (Cat 1, Cat 2 and Cat no) according to UN-GHS recommendations.
• Table 7 Results of the analysis of the solid products tested by the eye irritation test (IOT) and comparison with the results obtained by the Draize test
• Table 8 Measurement of the prediction ability of the eye irritation test to differentiate eye irritants (classified as Cat 1 or Cat 2) from non-irritants (Cat no) on a set of 15 solid chemicals
• Table 9 Confusion matrix analysis of the prediction ability of the ocular irritation test to be separated between 3 classes of irritation (Cat 1, Cat 2 and Cat no) according to the UN-GHS recommendations of the 15 solid products.

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